Voltage-gated potassium channels that are activated below the threshold for action potential initiation have a profound influence on neuronal excitability. Several diverse gene families encode "sub-threshold" potassium channels, but the precise contributions of many of these channels to neuronal physiology is poorly understood, primarily due to a lack of genetically-targeted animal models and specific small molecule modulators. Among the least characterized are Elk family potassium channels (KCNH3, KCNH4 and KCNH8) which have no distinguishing pharmacology, but are activated at typical neuronal resting potentials and are widely expressed in the nervous system. For example, Elk2, or KCNH3, is localized to the soma and dendrites of a variety of key principal neurons including pyramidal cells in the cerebral cortex and hippocampus and cerebellar Purkinje cells. It is associated with perisomatic GABAergic synapses, suggesting a role in inhibitory transmission. Specific small molecule modulators of Elk channels would be extremely important tools for characterizing the role of these channels in regulating neuronal excitability. Elk channel modulators would have the potential for development into therapeutics for diseases such as epilepsy which frequently involve hyperexcitability of the cortex and hippocampus. We have developed cell lines with inducible expression of Elk channels that are suitable for both fluorescent reporter-based HTS and patch clamp assays. These cell lines will be used to develop HTS assays for the identification of Elk-specific agonists and antagonists. Specific Aim 1 will focus on determining which reporter systems, voltage dependent dyes and ion-selective indicators, are best suited to reporting Elk channel activity in HTS- compatible formats. Specific Aim 2 will focus on optimizing assay parameters for HTS, testing the assay format with small-scale pilot screens and developing secondary assays for detailed evaluation of HTS hits. Relevance: A wide variety of brain diseases including epilepsy, anxiety and psychotic disorders involve pathological hyperexcitability of nervous system. Modulators of ion channels, which are the key mediators of neuronal excitability, have proven highly useful for treating these diseases. The research proposed in this application aims to develop a novel class of therapeutically relevant ion channel modulators in the hope that they will help patients that do not respond to current medications.